Currently, gene expression analysis can be quite challenging for heterogenous tissues such as tumors. The tumor microenvironment (TME) is a network of complex interactions between the tumor cells and surrounding immune cells. To identify new predictive biomarkers, it is essential to comprehensively characterize the immune cells within the TME at the molecular level. Traditional methods to assess gene expression in tissues lack either spatial information or sensitivity/specificity.
The ACD RNAscope™ in situ hybridization technology combined with the GeoMx® Digital Spatial Profiler (DSP) offers a powerful workflow for highly multiplexed, spatially resolved gene expression analysis. The combined workflow allows users to visualize up to three RNA targets using the RNAscope assay and to identify ‘regions of interest’ (ROI) for further interrogation by GeoMx DSP.
Our Joint Workflow Solution
- The power of single molecule visualization with RNAscope combined with high-plex gene expression analysis using GeoMx DSP RNA assays.
- Validated protocol with qualified RNAscope probes
- Molecularly guided high-plex spatial analyses on GeoMx DSP with single cell resolution
- Integrated manual and automated workflows
- Fresh frozen and formalin-fixed paraffin-embedded sample compatible
Technical Overview
- Visualize mRNA targets such as cytokines, transcription factors, checkpoint markers and immune cell markers using RNAscope for ROI selection
- Perform GeoMx DSP for targeted genes on selected ROI
- Quantify expression for 1,800+ gene targets in each region of interest with the GeoMx Cancer Transcriptome Atlas
- To see a detailed example, refer to the Tech Note ‘Resources’ section below.
How to Select Qualified RNAscope Probe Combinations
1.Identify study of interest (e.g. immune checkpoint)
2.Choose from qualified RNAscope probe combinations ( automated or manual ) from the table below or build your own for ROI selection to detect immune cells, tumor cells and soluble factors.
3.Order RNAscope Probes from ACD
Qualified RNAscope Probe Combinations Ordering Information
| Study of interest | Channel 1 Target | Channel 2 Target | Channel 3 Target | For Automated Probes | For Manual Probes |
|---|---|---|---|---|---|
| Immune checkpoints | PDCD1 (PD-1) | CD274 (PD-L1) | CD3E | Order Information | Order Information |
| Tumor vs. Stroma | panCK | PTPRC (CD45) | CD3E | Order Information | Order Information |
| Cytotxic T Lymphocytes (Set 1) | CD8A | GZMB | CD3E | Order Information | Order Information |
| Cytotxic T Lymphocytes (Set 2) | CD8A | IFNG | - | Order Information | Order Information |
| T regs | CD4 | FOXP3 | - | Order Information | Order Information |
| Activated T regs | CD4 | FOXP3 | TIGIT | Order Information | Order Information |
| Immune suppression | TGFB1 | FOXP3 | - | Order Information | Order Information |
| Macrophages | CD68 | CD163 | CD3E | Order Information | Order Information |
| M1 Macrophages | CD68 | CXCL10 | - | Order Information | Order Information |
| M2 Macrophages | CD163 | CCL22 | CD3E | Order Information | Order Information |
Resources
Recorded Webinar
Incorporating spatial genomics into your research using the RNAscope technology
Chimeric antigen receptor T (CAR-T) cells are promising treatments for hematologic and solid malignancies. CAR-T cells can recognize and eliminate neoplastic cells expressing specific protein antigens. However, the current generation of CAR-T cells cannot distinguish between neoplastic and normal cells that may also be expressing the same antigen, thus potentially resulting in “on-target/off-tumor” toxicity. In this GEN webinar, we will review the RNAscope™ in situ hybridization (ISH) method and show applications of the technology in the CAR-T cell and immuno-oncology fields. We will demonstrate how RNAscope ISH can be utilized to identify novel CAR-T cell targets and subsequently qualify monoclonal antibodies directed against those targets for immunohistochemistry. We will show how ISH can be used to predict CAR-T cell target organ toxicity in preclinical models. Finally, we will discuss optimal preparation of tissues, cell pellets, and cytospin slides for evaluation by ISH. You Will Learn - How RNAscope ISH technology can streamline your target identification and selection workflows - The advantages of using ISH for immunotherapeutic target cell selection - How ISH evaluation of target expression in normal tissues can be used to predict CAR-T cell induced toxicity - Optimal tissue, cell pellet, and cytospin slide preparation for RNAscope ISH
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